Gram quantities of 85-100% pure enzymically-active human alpha-thrombin (Fenton et al., 1977. J. Biol. Chem. 252:3587-3598) are being produced to enable detailed investigations on the structure, enzymic specificities, and biologic functions of this central enzyme in hemostatic and related physiologic processes. The 3-chain beta- and 4-chain gamma-thrombins, which retain certain enzymic activities but less than 1% the coagulant activity of the parent 2-chain alpha-thrombin, are also being prepared for parallel studies. Our studies include: (1) further development of preparative methods (e.g., autolytic forms, human Factors IX(a), X(a), etc., other species); (2) new analytical techniques (e.g., nephelometric fibrin detection, amido potentiometry, etc.); (3) reference thrombins (e.g., stable molar activities); (4) thrombin solute/solvent stability (e.g., denaturation vs. autolysis in salts, urea, etc.); (5) selective modification (e.g., mesyl-thrombin, carbohydrate derivatization); and (6) characterization of nonclotting thrombins (e.g., chromogenic substrates, active site probes, protein binding). Major studies with others include: (1) reference thrombin and products (Aronson/Bethesda); (2) sequence location of beta- and gamma-thrombin cleavage sites (Walz/Detroit); (3) thrombin crystallography (Stroud/San francisco); (4) exo-site affinity labeling (Bing/Boston); (5) spin-labeling and fluorescent-probing (Berliner/Columbus); (6) denaturation CD/ORD spectra (Fareed/Maywood); (7) antithrombin III/heparin reactions (Feinman/Brooklyn; Bang/Indianapolic); (8) factor VIII reactions (Gralnick/Bethesda); (9) platelet reactions (Detwiler/Brooklyn; Zimmerman/La Jolla; Mohammad/Pawtucket); (10) complement reactions (Bing/Boston; Hugli/La Jolla); (11) mitogenic effects (Buchanan/Cambridge; Gospodarowicz/San Francisco; Cunningham/Irvine); and (12) physiologic disposition (Jaffe/Bethesda). Our preparations have been (are being) used by 75-100 laboratories.